Production of streptomycin by culture immune to phage



PRODUCTION OF STREPTOMYCIN BY CULTURE IMlVIUNE TO PHAGE Fernando Carvajal, Ridgewood, N. J assignor to Schenley Industries, Inc., New York, N. Y., a corporation of Delaware Application December 20, 1954, Serial No. 476,241 8 Claims. or. 19s s The present invention. relates to improvements in the production of streptomycin by the submerged aerobic cultivationof selected strains of the organism Streptomyces griseus in an essentiallysynthetic nutrient medium comprising dextrose and dextrin in combination as a carbohydrate source.

The organism Strptomyces griseus is commonly cultivated in nutrient media containing carbohydrates and natural proteins to produce streptomycin. The yields produced in known processes generally vary from batch to batch, the recovery of the streptomycin is often complicated by the presence of complex metabolic products of the natural proteins, and the growth of the organism is sometimes inhibited by the presence of bacteriophage. Other disadvantages of the so-called natural nutrient media used for streptomycin production are the excessive foaming that may occur during fermentation and the difficulty of sterilizing such natural proteins, especially soybean meal.

In accordance which are readily available in purified or crystalline form, and whose only or principal source of carbohydrates is a mixture of dextrose and dextrin in certain definite proportions which are specified hereinafter. Although such a medium is not suitable for the growth of all strains of Streptomycesgriseus with the resulting production of high; yields of streptomycin, I found that a mutant strain of Streptomyces griseus, which was designated U76, of which a culture identified at ATCC 12475 was deposited with. the American Type Culture Collection, ,2029 M Street, N. W., Washington 6, D. C., and which isremarkably immune: to the action of bacteriophage frequently encountered in the production of streptomycin by these general methods, grows in such a medium and produces yields of streptomycin-"as high as 3000 or more micro 2,808,364 Patented Oct. 1,1957

Grams per liter Ammonium sulfate (NH4)2SO4 10.0

Sodium nitrate, NaNOa 2.0 Carbohydrates as follows:

Dextrose to 90% of total, dextrin 10% to 30% of total 40 to 80 Dibasic potassium phosphate, K2HPO4 1.0 Sodium chloride, NaCl 3.0 Magnesium sulfate, MgSO4.7H2O 02 Ferrous sulfate, FeSO4.7H2O 0.03 Zinc sulfate, ZnSO4.7H2O 0.03 Manganous sulfate, MnSOrAHzO Calcium carbonate, CaCOa 10.0 Distilled water to 1 liter.

Before the addition of the calcium carbonate, the medium is adjusted to a pH in the range of 7.5 to 8.0 (preferably 7.5). The calcium carbonate is then added and the medium is sterilized in the conventional manner. After sterilization, the pH of the medium falls within the range 6.5 to 7.0. The production of streptomycin with this medium otherwise contemplates no changes in the process of cultivating a strain of Streptomyces griseus in the medium at prevailing room temperatures, or preferably between and F. (24 and 27 C.), and recovery of the streptomycin by conventional methods.

In several experiments, a total of 16 shake flasks using the above preferred synthetic medium gave an average yield of 3700 ,ug. per ml. with a range from 3000 ,ug. per ml. to 4300 g. per ml. The carbohydrate concentrations in these experiments were 40 grams of dextrose and 10 grams of dextrin per liter. These results are typical of those obtained in numerous other shake-flask experirnents.

Instead of the mixture of ammonium sulfate and sodium nitrate which is specified in the foregoing preferred medium, other simple nontoxic ammonium salts and ni trates may be used. Thus, for example, a mixture of ammonium phosphate and potassium nitrate may be used, in which event it may be desirable to compensate for the phosphate ion thus added by reducing the quantity of potassium phosphate in the medium. Likewise, other phosphate salts, for example, sodium phosphate, may be used to replace potassium phosphate providing the potassium ion concentration is brought to a suitable level by addition of other potassium salts. The proportions of the constituents of the medium other than the carbohydrates maybe varied generally within the ranges used in media of this character, but the carbohydrate content should be maintained in excess of approximately 4% and less than approximately 8% and preferably between 5 and"6%, .and should consist, as specified, of a mixture of. from approximately 70' to approximately 10 to 30%"of dextrin, and preferably a mixture containing 75 10.85% of dextrose and'lS to 25% of dextrin.

Although I specify'a dextrin and dextrose as the sources of carbohydrates, the dextrosernay be replaced, eitherand other disaccharides that Enzymes forme'dduring 5 the growth of the. organism promote such hydrolysis ofstarches which have been wholly or in part, by sucrose yield dextrose by hydrolysis.-

polysaccharides. Dextrins are converted by torrefaction, either with or without acid or base catalysts, or by enzymatic action, to polysaccharides of lower molecular weight. Within the scope of this term are to beunderstood to be included such'starch products 7 as the so-called soluble starches and the commercialdex-g ittW of dextrose and from i trins which are sold as such or under various trade-mark designations, and which are referred to generally as watersoluble dextrins.

In another embodiment of my invention, small amounts of natural proteins are added to the foregoing preferred 5 synthetic medium. A preferred addition is approximately 1 gram of corn-steep liquor per liter of medium. Other natural proteins such as soybean meal and peanut meal may be used, either separately. or in combination with 1 each other. It'shouldbe emphasized'that'these additions o f'natural proteins'are 'not'essential to high streptomycin yields but may be used if des'iredffor other purposes, such as speeding up production. Also, the addition of these small amounts'tup to 10igra'ms per liter of medium) of'natural'protei'n's does not impair the many advantages of'thesynth'etic'nrediu'm. As examples of this embodiment, the following "yields of streptomycin were obtained in the pilot plantusi'ngth'e U-76 strain of Streptomyces gri's'euS' (ATCC-12475) and the foregoing preferred synthetic medium containing in addition 1.0 gram per liter ofcorn-st'eep liquor: 2600 ug. per ml. in 96 hours 3100 ,ug. per ml.-in 120 hours Sirnilar'high yields, as follows, were obtained when 1.0. gram of soybean meal was added to the preferred synthetic medium:

3 000 ,ugper ml. in 96 hours In still another embodiment of my invention, the addition of approximately 10 grams of corn-steep liquor per liter to the preferred synthetic medium has been used. Even with this high level of natural protein substances, some of the advantages of the synthetic media are retained. For example, there is still much less foaming during fermentation than with the wholly-natural medium, and, in addition, the difficulty of sterilizing large amounts of soybean meal is circumvented. As examples of this 40 embodiment, the following yields of streptomycin, indicative of the constancy of the results, were obtained in' individual pilot-plant runs:

3500 g. per ml. in 120 hours 3700 g.1per ml. in 120 hours 3900 g. per'ml. in 120 hours 3400 ,ug. per ml. in 96 hours It will 'be obvious to those skilled in theart that conventional variations may be made in the synthetic medium and that various natural proteins may if desired be added. to the synthetic medium, either separately or in combination, and at various levels. It is to be understood that my invention is not restricted or limited in any way by the above examples'and embodiments, alljof which-are given merely for purposes of illustrating my'invention.

The U-76 strain of Streptomyces griseus (ATI'C 12475) is a bacteriophage-immunestrain of'this organism which originated from a sample of Ohio river mud collected at Lawrenceburg, Indiana, in March 1945. A single colony grown from an ultraviolet-irradiated spore suspension of that culture, which was plated out on an agar medium containing 250 micrograms of streptomycin per milliliter, was isolated. A suspension of the spores of a--culture from that isolate was subsequently irradiated with'ultraviolet light and another single colony was plated from-an agar medium containing 250 micrograms per milliliter ofstreptomycin. A suspension of spores grown "from this colony was then mixed with afiltrate of a bacteriophage-active liquid and these spores were cultid inia liquid medium and subsequentlyplated outzon containing-250 micrograms of streptomycin per-milfrom.which;airphage+immune 1 colony was selected.

'Tbiinhu'ifirstrain was obtained y p agar containing "micrograms of streptomycin per milliliter 7 a spore suspension of this phage-immune colony that had been again irradiated with ultraviolet light.

Although I prefer to use this bacteriophage-immune U- 7 6 strain and its descendants and mutants with the medium described herein to obtain the specified high streptomycin yields, obviously other closely related strains and mutants, whether or not they are susceptible to bacteriophage degradation, may also thrive in this medium and produce high streptomycin yields.

As examples of the ability of the U-76 strain and its descendants and mutants to produce superior streptomycin yields in synthetic media, comparisons were made with another strain of Streptomyces griseus designated as 8-1 and previously used in-streptomycin production. The

average peak yields obtained in 11 shake flasks were compared, using these two strains and the same preferred synthetic medium. The following peak yield results were obtained:

g. per m1. U-76 strain of S. griseus 3200 8-1 strain of S. gr 1200 In each case the mediumcontained 1.0 gram of cornsteep liquor per liter.

To illustrate preferred methods of practicing the process ofmy invention and of the improved results obtained;v comparative experiments were conducted with the preferred medium specified hereinabove, but containing in addition 1.0 gram perliter of corn-steep liquor (penicillin grade containing approximately 50% by weight of nonvolatile solids), the dextose-dextrin ratios of which were varied while the total carbohydrate concentrations weremaintained at respectively 4%, 5%, 6%, 7% and 8% The U-76 strain of Streptomyces griseus (ATCCv 12475) was used and the dextrin was A. E. Staley & Co. Staclipse No. 1. Each of these experiments was performed in triplicate in shake flasks and the maximum (peak) potency obtained in each was determined. The

streptomycin content was assayed periodically by'the conventional microbiological assay method using Staphylococcus aureus ATCC 9996 as the standard organism.

The averages of the triplicates of each experiment that- These results are were thus obtained were as follows. plotted in the graph constituting the'drawing accompanymg this application.

Efiect of dextrose-dextrin ratio on streptomycin yield Dextrose Dextrtn' Peak (Percent (Percent: Yields by Wt.) by Wt.) g/m1.)

a a: a Total Oarbobydrates,4%.' 2.0 2.0 0.8 3.2 1, 610 0.0 4.0 1, 780 2'8 2'8 2118 I Tptal Oarb0hydrates,.5% 2. 5 2.5 21020 1. 0 4. 0 1, 910 0.0 5. 0 1, 865 6. 0 0. 0 480. 2'3 18 ea: Total Carbohydrates, 6% 3. 0 3. 0 11940 2. 0 4. 0 1, 950 1.0 5. 0 '2, 055 0.0 e. 0 2,190 7.0 0.0 1,000 6.0 1.0 1, 910 Y 5. 0 2.0 2, 090 Total Carbohydrates, 1% $3 :8 t. 3,33

- l 2.0 ;5. o 1, 680 L0 6.0 1,580 no "2.10 1,660 8. 0 0. 0 .890 7. 0 1. 0' 1,795 6.0 2.0 2,340 n m a. o 3.0. 1,830 Total Carbohydrates, 8% 4. 0 4. 0 l, 820 V 3.0. 5. 0 1, 700

Inasmuch as the foregoing specification comprises preferred embodiments of my invention, it is to be understood that the invention is not restricted thereto, and that modifications and variations in conventional manner may be made therein without departing from the invention, which is limited only by the scope of the appended claims.

I claim:

1. A process for the production of streptomycin which comprises the aerobic cultivation of the ATCC 12475 streptomycin-producing strain of the organism Streptomyces griseus in a nutrient medium comprising nontoxic ammonium and nitrate salts as the principal nitrogen sources and, as the carbohydrate source, between approximately 4 and approximately 8% by weight/volume of a mixture of a water-soluble dextrin and a sacchan'de of the group consisting of dextrose and disaccharides that are hydrolyzable to dextrose, the dextrin content of the carbohydrate source constituting between approximately 10 and approximately 30% 'by weight of the mixture, and subsequently recovering the streptomycin from the resulting broth.

2. A process as defined in claim 1 in which the total carbohydrate content of the medium is between approximately 5 and approximately 6% by weight/volume and the dextrin content of the carbohydrate mixture is between approximately and approximately by weight.

3. A process as defined in claim 1 in which the total carbohydrate source consists of between approximately 5 and approximately 6% by weight/volume of the medium, and consists of a mixture of Water-soluble dextrin and dextrose, of which the dextrin consists of between 10 and by weight of the mixture.

4. A process as defined in claim 1 in which the ammonium salt is ammonium sulfate and the nitrate salt is sodium nitrate.

5. A process for the production of streptomycin which comprises the aerobic cultivation of the streptomycin- 6 producing bacteriophage-immune ATTC 12475 strain of Streptomyces griseus in an aqueous nutrient medium comprising the following substances in approximately the following proportions specified in grams per liter:

Grams per liter Ammonium sulfate 10.0 Sodium nitrate 2.0 Carbohydrates to Potassium phosphate 1.0 Sodium chloride 3.0

also comprises soybean meal in a proportion between approximately 1 and approximately 10 grams per liter.

8. A process as defined in claim 5 in which the medium also comprises approximately 1 gram per liter of cornsteep liquor.

References Cited in the file of this patent UNITED STATES PATENTS 2,516,682 Donovick July 25, 1950 FOREIGN PATENTS 635,529 Great Britain Apr. 12, 1950 OTHER REFERENCES La Manna et 211.: Basic Bacteriology, 1953, Williams and Wilkins, pages 28-29. 

1. A PROCESS FOR THE PRODUCTION OF STREPTOMYCIN WHICH COMPRISES THE AEROBIC CULTIVATION OF THE ATCC 12475 STREPTOMYCIN-PRODUCING STTRAIN OF THE ORGANISM STREPTOMYCES GRISEUS IN A NUTRIENT MEDIUM COMPRISING NONTOXIC AMMONIUM AND NITRATE SALTS AS THE PRICIPAL NITROGEN SOURCES AND AS THE CARBOHYDEATE SOURCE, BETWEEN APPROXIMATELY 4 AND APPROXIMATELY 8% BY WEIGHT/VOLUME OF A MIXTURE OF A WATER-SOLUBLE DEXTRIN AND A SACCHARIDE OF THE GROUP CONSISTING OF DEXTROSEAND DISCCHARIDES THAT ARE HYDROLYZABLE TO DEXTROSE, THE DEXTRIN CONTENT OF THE CARBOHYDRATE SOURCE CONSTITUTING BETWEEN APPROXIMATELY 10 AND APPROXIMATELY 30% BY WEIGHT OF THE MIXTURE, AND SUBSEQUENTLY RECOVERING THE STREPTOMYCIA FROM THE RESULTING BROTH. 